Production of harringtonine and isoharringtonine

ABSTRACT

Alkaloids found to be chemotherapeutically active against certain strains of leukemia in mice are produced from Cephalotaxus harringtonia. The process includes extraction of plant parts with a polar solvent, partitioning the plant extracts between nonpolar and acidic aqueous solvents, making the aqueous portion basic, and removing the crude alkaloids from the basic aqueous solution. The crude alkaloids are purified and used in the treatment of leukemic mice.

United States Patent 1 Powell et a1.

PRODUCTION OF HARRINGTONINE AND ISOHARRINGTONINE Inventors: Richard C.Powell, Peoria; Cecil R.

Smith, .lr., Dunlap, both of Ill.

OTHER PUBLICATIONS Powell. et 111., Abstract No. 33. Abstracts ofPapers, 162ml Nat. Meeting, American Chemical Society, Wash, D.C., Sept.l217, 1971.

Powell, et al.. Chemical Abstract, 721121749 (1970). CCNSC. CancerChemotherapy Reports, No. 25, Dec. 1962, Protocol 1.3033LE.. pp. 3,12.52.

National Cancer Institute, Drug Research and Development, Chemotherapy,Screening Data Summary 1 1 Mar. 11, 1975 Interpretation Instruction 14.March 1972. Bethesda. Md.

Powell, et al., Tetrahedron Letters. 46. 4081-4084. (Oct. 1969), QD241.T42.

Abraham, et al.. Tetrahedron Letters. 46. 4085-4086. (1969), QD 241.T42;Chem. Abstract 71:1296942. Gilbert, et al., .1. Am. Chem. Soc., 86.694-696. 1964. QD1.A 5.

Powell, et al., Abstracts of papers. 158th National ACS- Meeting. NewYork. Sept. 7-12. 1969. MEDI- O6I.

Primary E.taminerDonald G. Daus Assistant E.\'uminerRaIph D. McCloudAttorney, Agent, or FirmM. Howard Silverstein; Max D. Hensley; David G.McConnell [57] ABSTRACT Alkaloids found to be chemotherapeuticallyactive against certain strains of leukemia in mice are produced fromCepltalotmus ltarringtonia. The process includes extraction of plantparts with a 'polar solvent. partitioning the plant extracts betweennonpolar and acidic aqueous solvents, making the aqueous portion basic,and removing the crude alkaloids from the basic aqueous solution. Thecrude alkaloids are purified and used in the treatment of leukemic mice.

3 Claims, 3 Drawing Figures DEFATTED MEAL OR 4 4 GROUND PLANT PARTSWATER OR ALC OI-IOL EXTRACTION PART! TIONING CHCI3-TARTARIC ACIDSOLUTION TARTAFQIC ACID SOLUTION 5 LIRESIDUE I 6 ADDITION OF BASE NDEXTRACTION BY RESIDUE CHCI CRUDE ALKALOID A/ 9 EXTRACT PATENTEUHRR n masum 1 or 2 SEE D I KERNEL FIGJ' OF OIL EXTRACTIO DEFATTED MEAL DEFATTEDMEAL OR 4 GROUND PLANT PAR WATER OR ALCOHOL EXTRACTI PART! TIONING cHClaRTARIE ACTD RESIDug I 6 SOLUTION TARTARIC ACID [EXTRAQTI 7 SOLUTION a 8IO ADDITION OF BASE Fl 6 2 ND EXTRACTION BY \ESIDUE 0 CHCI-3 CRUDEALKALOID 9 EXTRAC 1 PRODUCTION OF HARRINGTONINE AND ISOHARRINGTONINE Anonexclusive, irrevocable, royalty-free license in the invention hereindescribed, throughout the world for all purposes of the United StatesGovernment, with the power to grant sublicenses for such purposes, ishereby granted to the Government of the United States of America.

BACKGROUND OF THE INVENTION This is a continuationin-part of applicationSer. No. 26,995, filed, Apr. 9, 1970 now abandoned.

This invention relates to two novel alkaloids, harringtonine andisoharringtonine, and has their production as its primary object. Asecondary object is to provide chemotherapeutic agents for the remissionof leukemic tumors. Leukemic mice treated with these compounds show aremarkable increase in survival time over untreated leukemic mice. I

The search for compounds that are useful in cancer chemotherapy has beenvigorous but positive results have been relatively few [Chem Eng. News44(51): 64-68 (1966); Agr. Res, July 1966, pp. 3-4; Amer. Hort. Mag. 47:336-337 (1968)].

Alkaloids have been found in several species of Cephalotaxus, and it hasbeen shown that cephalotaxine is the major alkaloid in most of thespecies studied [Paudler et al., J. Org. Chem. 28: 2194-2197 (1963)].However, cephalotaxine has been shown to be inactive toward the L1210and P388 strains of leukemia.

Briefly, in accordance with the invention, the inventors producedchemotherapeutically active alkaloids from C. harringtonia by thefollowing procedure:

a. Ground defatted seed or other plant parts are extracted with 95percent alcohol or its equivalent in order to obtain a solution of thedesired materials.

b. The extract solution is then concentrated to about 5 percent of theoriginal volume.

c. The concentrated extract solution is then partitioned between anapproximately 1 to 1 mixture of chloroform and 6 percent aq. tartaricacid, and the two layers are separated.

d. Base is added to the tartaric acid solution to change the pH to about9 as measured by pH paper.

e. The basic aqueous solution is then extracted with chloroform, and acrude alkaloid mixture is ob tained.

f. The crude mixture of alkaloids is subjected to a series of separationprocedures which include countercurrent distribution (CCD), thin-layerchromatography (TLC), and column chromatography (CC). These proceduresresult in the collection of several fractions, each of which willcontain from one to several alkaloids.

g. The final isolation step is to purify the individual compound, i.e.,harringtonine or isoharringtonine, by TLC.

The preferred separation is to start with a CCD of from 10 to 200individual extractions in a chloroformpH 5 aqueous buffer solventsystem, followed by either preparative TLC or CC. Harringtonine andisoharringtonine are both easily purified by TLC.

The secondary objects, remission of leukemic tumors in mice. isaccomplished by intraperitoneal injections of harringtoninc orisoharringtonine in saline or its equivalent. Several routes oftreatment were tried and the results were tabulated (see Example 3). Thepreferred route was to inject the mouse, starting 24 hours after tumorimplant, once a day for 9 days.-

The optimum daily dosage for harringtonine against Ll2l0 was determinedto be from 0.2 to 1 mg. of alkaloid per kilogram of mouse body weight.In one test, a 2-mg./kg. dose proved to be toxic. A lmg./kg. dose gavethe highest survival time. Harringtoninc administered in a total dosageof from 2 to 12 mg./kg. of mouse weight over a period of from 1 to 9days was effective against leukemia strain P388. and 2 milligrams perday gave the same survival time without toxic effect. Dosages forisoharringtonine were 5 to 10 mg./kg. per day against leukemia strainLl2l0 and 3 to 12 mgjkg. per day against strain P388 with no toxiceffect.

The above-stated objects will be further clarified in the detaileddescription and the examples to follow.

DETAILED DESCRIPTION OF THE INVENTION FIGS. 1, 2, and 3 of theaccompanying drawings are flow diagrams or fractionation treesdescribing the method of obtaining chemotherapeutically active alkaloidcompounds from the seed or plant parts of Cephalotaxus harringtonia. C.harringronia is native to the mountain forests of Japan, from centralHonshu to Skikoku and Kyushu. It is also cultivated in the UnitedStates, Europe, and Japan (Robert E. Perdue, Jr. et al., The AmericanHorticultural Magazine, Winter 1970, pp. 19-22).

Starting with a quantity of seed 1, FIG. 1, the hull is removed bystandard methods leaving ground seed kernel 2 from which the oil isextracted with a nonpolar solvent. The seed oil 3, which is notpertinent to the instant invention, was stored for future use. Drieddefatted meal or ground plant parts 4, FIG. 2, are subjected toextraction with a polar solvent (i.e., water, alcohol, mixtures of waterand alcohol, or any equivalent solvent which will dissolve thealkaloids) yielding extract 5 and residue 6. Residue 6 is discarded andextract 5 is concentrated to approximately 5 percent of its originalvolume and partitioned between two immiscible solvents, one of whichmust be an acidic aqueous solution and the other a polar solvent capableof dissolving the material not soluble in the aqueous acid layer.However, the preferred partitioning system is chloroform and 6 percentaqueous tartaric acid solution.

which has a pH of about 2. Any acidic solution would be suitable forthis extraction. However, the alkaloids are labile to low pH, e.g.,mineral acid, so an organic acid such as tartaric or citric ispreferred. Materials collected from the chloroform extract 7 contain nobasic alkaloids and are discarded. A sufficient amount of base is addedto the tartaric acid solution 8 to increase the pH to 9-10, asdetermined by pH paper. Ammonia or Na CO is preferred because of costand convenience. When the crude alkaloids dissolved in the aqueous acidsolvent of the partitioning system are made basic, they become solublein chloroform which is used to extract them from the now basic aqueoussolution. The chloroform solution is dried and evaporated to yieldextract 9, a chemotherapeutically active crude mixture of basicalkaloids.

Separation and purification of the alkaloids, barringtonine andisoharringtoninc, could be accomplished by any of innumerablecombinations of (7CD, CC, and TLC. Either ofthe alkaloids could beisolated from the crude mixture by TLC alone. However, this would bevery inefficient as it would necessitate a large plurality of TLC platesto obtain a useful amount of any individual alkaloid.

tained on a known sample of pure eaphalotaxinc, Tables3-7.

The problem of efficiently obtaining the individual Table 1 alkaloidswas solved by a preliminary separation by 5 CCD, FIG. 3, having achloroform lower phase and an Done Leukemia Survival time upper phaseconsisting of pH 5, Mcllvaines standard Alkaloid mg./kg. strain (T/C)buffer solution (Handbook of Chemistry and Physics, 9 20 U210 127 39thEdition, Chemical Rubber Co., Cleveland, Ohio, 50 U210 103 p. 1615).Countercurrent distribution is preferred be- 10 cause it is capable offractionating much larger samples 18 P388 147 than any of the othermethods. 19 50 L1210 112 The pH of the buffer is rather critical. If theaqueous 20 5 U210 2| 25 U210 88 solution of the CCD is more acidlc thanabout pH 4, all 22 25 U210 94 components move together with the buffer,and if the l3 6 U210 129 13 6 P388 147 pH is more alkaline than pH 6,there IS essentially no movement of the samples components with theaqueous phase and no separation.

Using analytical TLC as a guide, the individual'frac- Table 2 tions fromthe CCD of the crude alkaloid mixture are combined to give 11, 12, and13, each of which are Mk I d lk r 10ml l mixtures of two distinct (byTLC) compounds; extract a llmds mum! 15, a mixture of at least fivecompounds; and extract 16 43 14, which is essentially a single alkaloid.Further sepa- :3

ration and purification is accomplished by either prepa- 2O 4 ration TLCor CC. Essentially pure alkaloids l6, 17, Others 22 l8, l9, and 20 areobtained by preparative TLC of 11, I

Analysis is from one access-non. 5 and C umn Chromatography fractlonatesPercentages vary somewhat from one accession to another.

Table 3 Compound lo ('ephalotaxine 8(p.p.m.) Multiplicity J (H7)5(p.p.m.) Multiplicity .lt H/J 6.65 1 (1.03 l 1 616i 1 6.60 1 5.86 15.90 1 4.89 1 4.85 1 4.70 2 9.2 4.71 2 9 3.70 1 3.67 1 3.63 2 9.2 3.02 2J into two separate mixtures ot'alkaloitls, 2] and 22. No Table 4further fractionation olextract 13 was attempted.

Chemotherapeutic activity of each compound or Rclfllivc mixture, 13,l622, was determined in mice which 45 m/c l6 ('cfhilllmnc were implantedwith either of two types of leukemia 28 cells, lymphoid or lymphocytic,strain Ll2l0 or P388, 4 10.5 3.6 respectively. Starting 24 hours afterthe tumor implang; tation, previously determined dosages of each com-110 l().8 125 pound are injected intraperitoneally once a day for 9days. Survival time of treated leukemic mice is com- 128 pared to thatof untreated leukemic mice (T/C X 100), 137

. 149 12.7 15.2 Table l. A survlval time of 100 percent mdicates no ac-150 233 270 tivity. A T/C of greater than 100 percent means that 161 9.010.4 the treated mice are surviving longer than the control 5? mice, andthe compound used for treatment is actively 228 1 1.7 1310 retarding theprogress of the cancer. 5;:

Table 1 shows that compounds 18 and 20 were active 266 9.1 1012 towardeach leukemia strain. Fraction 13, known to be a mixture, showedactivity which was found to be due 284 to the presence of compound 18.285 I483 Compounds 18 and 20 were named harrmgtonme 299 122 121 andisoharringtonine, respectively, and their structures 38? weredetermined. I i 5 2 The ma or alkalold of C. harrmgtoma, Table 2, was99.9

shown to be cephalotaxine by comparing the results of analysis by theinventors on their isolate 16 to those ob- All relative intensity valuesof about It) and above are included.

Compound 1 6 Anal. calcd.

C H N O OCH Cephalotaxine Anal. calcd. C

( m zi m MW 315): 68.5

Found:

Found:

Table 7 Compound 16 Wave length (1,01 7.00 7.05 7.37 7.40 7.51 7.627.14) K64 8. .25 1.42 was 0.80 10.12 10.70 1 1.10 1 1.45 1 1.70

X- 'l'ransmittance 70 34 37 46 8X 91 83 17 70 48 11 53 51 60 (73 48 4255 (\1 25 50 2X 32 2X NOTE: lRs of both samples are superimposable.

The partial structure assigned to eephalotaxine by Paudler et al.,supra, is:

Cephalotaxine 7r- Transmittancc NMR studies, Table 3, show that thecomplete structures must be either:

(Structures as drawn in this specification are not intended to indicatethe stereochemistry of the compounds they represent and should not limitany compound herein to a specific structural configuration.)

The NMR spectrum contains two singlets, each equal to one proton, at86.65 and 86.61. These peaks are assigned to para aromatic protonsadjacent to oxygen functions (H and H,,). A peak with an area equivalentto two protons is observed at 85.86 in the spectrum of cephalotaxine.This peak and an infrared band at 10.7 ,u., Table 7, are characteristicof protons in methylenedioxy groups attached to an aromatic ring. Thespectrum contains an olefinic proton resonance as a singlet at 84.89 (HThe absence of coupling means that there are no vicinal protons. A lowfield doublet ap pears at 84.70 (H and this proton is coupled to onewhose signal appears at 83.63 (H .1 9.2 Hz). Upon aeetylation ofcephalotaxine, the 84.70 doublet moves down field to 85.80, a change of1.10 ppm, which shows the proton is attached to the same carbon atom asa hydroxyl function. The position of the methoxyl resonance at 83.70shows that it is attached to an unsaturated carbon; this assignment issupported by a strong infrared band at 6.07 .1.. The final structure ofcephalotaxine was determined by single crystal X-ray diffraction studiesof cephalotaxine methiodide. The derivative was prepared by reacting0.303 grams of cephalotaxine with 6 ml. of methyliodide for 1 hour.After evaporating the reaction mixture to dryness, the white solidcephalotaxine methiodide, 0.47] grams. was recrystallized from methanolto give a crystalline material that analyzed as follows:

3,87 7 Anal. calcd. for C H NO I (MW 457): C, 49.90; H, 5.29; l, 27.75%.Found: C, 50.00; H, 5.51; l, 27.78%.

A single crystal analyzed by an automatic diffractometer shows latticeparameters of: a 9.225; b 11.456;

sponds to the molecular ion peak (C H NO M m/e 53]) minus (C H O Knownsamples of acetyl cephalotaxine and cephalotaxine also have a peak atm/e 298 which corresponds to (M* C H O and (M c 19.569 A and B 10036. Adensity of D, 1.53, 5 OH), respectively. Accumulated data from lR, UV,and determined y flotation, corresponds t four mo ecules MS indicatingthat compounds 18 and are esters of per unit cell. The above dataindicate a space group of cephalotaxine is further substantiated bycomparing 1/ NMR data from cephalotaxine with that from the twoIntensities from 1.729 independent reflections were compounds, Table 9.

Table 9 1 Compound ll H 11 II II 1 a s 6 8 Q OCH Cophnlotnxino 6.65 6.613. 63 4.70 o. 89 5.86 3. 70

Spin decoupling un altar structure 11 z The can contain used to obtainrelative structure factors. The sharpened, three-dimensional Pattersonsynthesis obtained from the normalized relative structure factorsplainly showed the position of the iodide molecules. A fused liveandseven-membered ring system is evident when a difference synthesis isplotted with the contribution from the iodine atoms removed. When thefused ring system is removed in a subsequent difference synthesis, thefinal structure of cephalotaxine is elucidated as being I.

STRUCTURE OF ALKALOIDS Compounds 16, 18, and 20 were subjected to IR,UV, NMR, and MS. Compound 16 has already been shown to be cephalotaxine.

Infrared spectra were obtained for each alkaloid in a chloroformsolution. Compounds 18 and 20 have essentially superimposable spectrawhich contain a fairly broad band due to hydroxyl at 2.80 p. with ashoulder at 2.70 p. whilecephalotaxine has a single sharp band at 2.75a. A strong band at 5.77 p. due to ester carbonyl in 16 is absent incephalotaxine, while all contain a strong band at about 9.6 p. and abroad band at 10.7 1.1. which are characteristic of amethylenedioxyphenyl grouping. Further evidence of themethylenedioxyphenyl group in alkaloids l8 and 20 is obtained bycomparing their UV spectra with that of cephalotaxine which is known tocontain this chromophore, Table 8.

Table 8 Mass spectroscopic analysis shows that compounds 18 and 20 havea base peak at m/e 298 which correed to verify luignmut.

H It etc rule: to I 301W.

1 second nothozy group in the 1! portion.

III

In order to characterize the ester portion of the alkaloids, they weretransmethylated in the following mannerf Table 10 Wt. Time 'lemperaVolume oi 0.5 M sodium Alkaloid (g.) (hr.) ture methoxide (ml) 18 0.254l reflux 5 20 0.253 2 50 C. 5

Ethyl ether (40 ml.) was added to each reaction mixture which was thenwashed four times with l0-ml. portions of 5 percent aqueous acetic acidand once with distilled water. The aqueous portion was made stronglybasic and extracted with chloroform. The remaining aqueous portion wasacidified and extracted with ethyl ether which was combined with theoriginal ether layer. EAch of the two chloroform extracts, 7075 percentof the total recovered weight, containing an alkaloid which was shown tobe cephalotaxine by the fact that IR, UV, NMR, and MS data from the twoalkaloids were identical with those of known sample of cephalotaxine. Asan illustrative example of this identity, UV and melting point data arerecorded in Table 11.

Table l l SOLII'CC fi l MP. C. mmr g 6) mi" g mvu' (log 6) ccphalotaxlnc(uncorr.) (m,u.) (my) i -l IX I35|36.5 290 (3.6l) 260 (2.73) 238 (3.54)31) 1354365 290 (3.62) 260 (1.65) 238 (3.5?) ephalotuxus lilo-I375 290(3.64) 260 (2.75) 238 (3.56)

For purposes of simplification, the dimethyl ester products from thetransmethylation of alkaloids l8 and 20 will henceforth be labeled 18-1and 20-1, respectively. Infrared analyses of these products show strongbands in the region assigned to hydroxyl groups.

Mass spectroscopy of 18-1, using an excessive sample pressure method,gave a (M* 1) peak at m/e 249 which responds to a (M*) ion mass ormolecular weight of 248 (C H O When it is considered that thetransmethylation added one methyl group to each molecular specie, the MSdata for 18-1 agrees exactly with MS data for alkaloid 18; i.e.,cephalotaxine esterified with 18-1 would give a compound having amolecular formula (C ,,H O and a MW of 531. Ester 20-1 did not give a(M* 1) peak. However, the largest MS peak from 20-1, m/e 189,corresponds to (M* 59) from a compound with a molecular weight of 248,having lost a (CO- CH fragment.

Table l2 Fragment Fragment m/e Q n U a 11 (CIUHLSOS) (CZHGOII) Thefractionation pattern of 20-1, Table 12, indicates that it has asomewhat different structure than 18-1.

The final assignment of structure for the two ester The total structurefor alkaloid l8, harringtonine, and alkaloid 20, isoharringtonine,having been established by spectral characteristics and synthetictransformation (see also Abstract No. 33, Abstracts of Papers, l62ndNational Meeting, American Chemical Society, Washington, DC, Sept.12-17, l97l are as follows:

where R equals H a (c1 -(m -%a1 -o-cu for harringtonine, and

(m -cn-( e1 g -Laur for isoharringtonine.

Table 13 Ester Assignment WM, (multiplicity) 20-1 u w (multiplicity)(CH3) z' (CH2) 2'5 2 5 (SHISJ EXAMPLE 1 Extraction of Alkaloids from C.harringwnia. Cephalotaxus harringmnia seeds, 990 grams, were put throughcracking rolls to loosen the hull from the kernel. A hand separation ofkernels from hulls yielded 547 grams of kernels which were then groundto finer particles so that a more facile extraction of oil could beachieved. After a few hours of extraction with pentanehexane solvent ina Soxhlet extractor followed by evaporation of the solvent, 27l grams ofoil were obtained. The oil-free meal was air dried and allowed to standovernight in 600 ml. of 95 percent ethanol, after which the meal wasfiltered and extracted more times with 300-ml. portions of 95 percentethanol. The combined ethanol extracts were concentrated to a volume of100 ml. and diluted with 1 liter of 6 percent aqueous tartaric acidsolution. Four 200-ml. portions of chloroform were used to wash theaqueous tartaric acid solution. After washing, the aqueous solution wasadjusted to pH 9 by adding solid sodium carbonate. Ten grams of a crudealkaloid mixture, 9, were obtained by extracting the basic solution fourtimes with 200 ml. chloroform and evaporating to dryness the combinedextracts. The crude alkaloid mixture was dissolved in 400 ml. of pH 5Mcllvaines buffer and 400 ml. of chloroform, all of which was dividedevenly between the first 10 tubes of a 200-tube countercurrentdistribution apparatus, Model No. C-3, H. 0. Post Scientific InstrumentCompany, Inc. Forty milliliters of chloroform lower phase were placed ineach of the remaining 190 tubes. The machine was set to automaticallydeliver 40 ml. of pH 5 buffer upper phase into the system at eachtransfer. shake for several minutes, then rest for 5 minutes. This cyclecontinued for 190 transfers. The contents of each tube were made basic,pH 9-10 by adding sodium carbonate, and extracted twice with chloroform.After the chloroform was dried and the residue weighed, the residueswere combined as follows:

Fraction 12 was spotted on five standard TLC plates prepared from silicagel l ml. thick and developed with 15 percent methanol in chloroform.The bands were visualized by covering all but 1 cm. along the platesedge with an l impervious covering and placing the covered plate in an lchamber. The position of the bands were marked and the exposed l-cm.strip discarded. Two bands were obtained from the five TLCs; the bandwhich traveled the farthest being labeled fraction (or alkaloid) 19, theother fraction (or alkaloid) l8.

Fraction II was subjected to the same TLC procedure as fraction 12 andresulted in the separation of alkaloids l6 and 17. Alkaloid was obtainedin the same manner from fraction 14. Fraction 15 was separated intofractions 2] and 22 on a column of 50 grams of a commercially availablesilicic acid by successively percolating through the column ml.chloroform, I80 ml. 25 percent methanol in chloroform, and 200 ml. 50percent methanol in chloroform. These two fractions, however, werechemotherapeutically inactive and were discarded. Fraction 13 wasactive, but

12 subsequent investigation showed that its activity was due to itspresence of 18.

EXAMPLE 2 ......="0= n 0.0 UN

Fractions a and b contained a mixture of alkaloids as analyzed by TLCbut were not examined further. The following table shows the methods ofpurification and yields of alkaloids from the remaining fractions.

Table 15 Method Pure alkaloids Fractions of (grams) purification l6l8 1) 20 c d e Prep. TLC 0.04 0.33 f Prep. TLC 0.10 0.22 0.02 g Prep.TLC a 0.27 0.60 h Prep. TLC 0.40 0.94 1 Col. Chromo. |.02 032 1 Col.Chromo. 2.76

TOTAL 3.78 0.8l 2.08 0.35

Same as Example 1. Column chromatograph carried out on I00 g. ofBroekman grade III neutral alumina, eluted with 350 ml. benzene.followed by 200 mlv ethyl ether.

Analysis of these four alkaloids by IR, NMR, MS, and GLC showed them tobe essentially pure compounds.

EXAMPLE 3 Mice implanted with either lymphoid leukemia' Ll2l0 orlymphocytic leukemia P388 were divided into groups of 10 for treatmentwith harringtonine. Similar groups of leukemic mice were left untreated,and the survival time (T/C) in days was computed. Treatment was given byintraperitoneal injection of harringtonine in saline solution. Theschedule of treatment and the results are given in Table 16.

03H every 3 hours; OD every day; 04D 7 every 4 days. elc. mg/kg;milligrams of alkaloid per kilogram of animal body weight.

3,870,727 13 14 The optimum scheduling (i.e., intraperitoneal injecwhereR equals tion once a day for 9 days) was used to treat mice implantedwith either leukemic strain Ll 210 or P388, and

survival time (T/C) was computed, Table 17. g B E (on (on ca O-(ZI Table17 3 2 3 Leukemia Dose, Alkaloid strain mgJkg. T/C,

or Harringtonine L12 [0 2 Toxic Ll2l0 1 I32 F510 8.; 122 H 3 1 10 12P388 2 147 l H -0-CH P388 1 147 P388 0.5 142 I P388 0.2 126 I5lsoharringtoninc U210 l 15 2. The alkaloid compound of claim 1 wherein RP388 I2 209 P388 6 l83 P388 3 139 H H rab-24 112)? HE-E- HQ We claim: 7l. The alkaloid compound having the following structure:

25 3. The alkaloid compound of claim 1 wherein R OCH R0 3 (CHa)2- 2)2- mt

1. THE ALKALOID COMPOUND HAVING THE FOLLOWING STRUCTURE:
 1. The alkaloid compound having the following structure:
 2. The alkaloid compound of claim 1 wherein R 